Because metastatic pancreatic cancer cells can disseminate from the primary tumor during the earliest stages of cancer progression, long before (10 years) the patient becomes symptomatic, the majority of patients have advanced stage metastatic disease at the time of diagnosis. This and the fact that patients resist all forms of conventional chemotherapy, pancreatic cancer has a dismal 5 year survival rate of less than 5%, and a median survival of 4-6 months. Clearly, to combat this devastating disease new biomarkers for early detection and new therapeutic targets directed at eliminating metastatic cells are urgently needed. Therefore, the objective of work in this proposal is to identify new proteins that mediate pancreatic ductal adenocarcinoma (PDAC), which can be used as biomarkers at and therapeutic targets to treat metastatic PDAC. To achieve this goal, in Aim 1 we will generate highly metastatic genetically engineered mouse and patient-derived PDAC cell lines by serial passaging of isolated metastatic cells in the pancreas of athymic nude mice. We will then biochemically purify their invadopodia, which drive PDAC cell invasion and metastasis, for proteomic and phosphoproteomic analyses. To identify metastatic signatures, the invasive invadopodia membranes will be interrogated for changes in total protein profile as well as kinase activation networks using established phosphosite identification methods, quantitative MudPIT mass spectrometry, and bioinformatics. In Aim 2, we will functionally test selected key signature proteins identified in Aim 1 for their ability to mediate PDAC cell metastasis using RNAi knockdown technology and preclinical animal models of PDAC cell metastasis. Our unique approach of using in vivo selected metastatic variants combined with invadopodia purification and comprehensive proteomics will reveal important new metastatic signature proteins that serve as 'fingerprints' to identify and treat metastatic PDAC cells. The identification of novel metastatic proteins is the first, and critical step, in the process that drives development of new PDAC therapeutics and diagnostics, which are sorely needed to treat this devastating disease.